The overall objective of the entire project is to elucidate mechanisms of heart looping. Earlier work in this laboratory has provided us with a testable hypothesis relating the ontogenic appearance of myofibrils in the myocardium (differentiation) to the generation of the loop. This proposal contains descriptive projects designed to permit us to map developing fibrils and relate them to heart shape and also to experimentally manipulate their orientation and alignment in situ. This will permit us to discern the effects of myofibrillar alignment on morphogenesis. Similar experiments will be done on cultured cells to relate myofibrillogenesis to cell shape. We shall also relate the total amount of cardiac muscle myosin in different regions of the developing heart, as determined by quantitative immunofluorescence, to shape generation. Coincidentally we shall examine early myocardial cells, before and just after phenotypic expression for the presence of "non-muscle" (defined immunologically) myosin. Since we have earlier shown that cell shape changes are important in heart looping, we will determine whether heart cells have a stress fiber system that contains non-muscle myosin before they develop myofibrils. Additional experiments will be done by ablating specific regions of the myocardium at early tubular stages to determine if any group of cells are crucial to the generation of the loop. We will also continue the studies involving the injection of enzymes into the cardiac jelly in an effort to understand the role this compartment plays in morphogenesis. We shall begin to explore an animal model of situs inversus using mice homozygous to gene IV. This proposal, therefore, represents a multi disciplinary study, both experimental and descriptive, of the regulation of early heart shape. As such, it should help us understand possible etiology of congenital heart disease.